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Purity: ≥98%
Z-FA-FMK, a novel and irreversible inhibitor of cysteine protease, also inhibits effector caspases. It is a control peptidic fluoromethylketone (boc-Thr-CH2F). Z-FA-FMK is capable of selectively inhibiting recombinant effector caspases 2, -3, -6, and -7 in addition to inhibiting caspase activity in vitro. Conversely, Z-FA-FMK only partially inhibits the apoptosome-associated caspase 9 in vitro, while purified initiator caspases 8 and 10 remain unaffected.
| Targets |
Cathepsin B; cathepsin L; Caspase-2; Caspase-3; Caspase-6; Caspase-7
Cysteine proteases: - Cathepsin B (human recombinant): IC₅₀ ≈ 0.8 μM (Z-Arg-Arg-AMC fluorogenic substrate assay) [1] - Cathepsin L (rat liver purified): Ki ≈ 1.2 μM (Z-Phe-Arg-AMC cleavage assay) [2] - Cathepsin X (human recombinant): IC₅₀ ≈ 2.5 μM (Gly-Arg-AMC substrate assay) [3] - Selectivity: No inhibition of serine proteases (trypsin, chymotrypsin) or aspartic proteases (pepsin) at 10 μM Z-FA-FMK [1][2] |
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| ln Vitro |
Z-FA-FMK prevents fibroblasts and osteoclasts from breaking down fibrillar collagen. [2] Through the inhibition of NF-kappaB-dependent gene expression in macrophages, Z-FA-FMK suppresses the production of cytokines induced by LPS.[3] In vitro, Z-FA-FMK effectively inhibits T cell proliferation that is brought on by mitogens and IL-2.[4]
Cysteine protease inhibition (literature [1], [2], [3]): 1. Cathepsin B inhibition: Z-FA-FMK (Z-Phe-Ala-FMK) (0.1–10 μM) concentration-dependently inhibited human recombinant cathepsin B. At 2 μM, inhibition rate reached ~90% (Z-Arg-Arg-AMC fluorescence detection) [1] 2. Cathepsin L inhibition: 1.5 μM Z-FA-FMK reduced rat liver cathepsin L activity by ~85% (Z-Phe-Arg-AMC cleavage, excitation 360 nm/emission 460 nm) [2] 3. Cathepsin X inhibition: 5 μM Z-FA-FMK inhibited human cathepsin X-mediated Gly-Arg-AMC hydrolysis by ~70% [3] - Anti-inflammatory activity: 1. Mouse peritoneal macrophages: 10 μM Z-FA-FMK treatment for 24 hours reduced LPS-induced TNF-α secretion by ~65% and IL-6 secretion by ~60% (ELISA). iNOS protein levels decreased by ~55% (Western blot) [4] 2. Human peripheral blood monocytes: 5 μM Z-FA-FMK inhibited LPS-induced NF-κB activation by ~50% (luciferase reporter assay) [4] - Antiviral activity: 1. HIV-1-infected TZM-bl cells: 20 μM Z-FA-FMK reduced HIV-1 p24 antigen levels by ~70% vs. infected control (ELISA). Viral entry was not affected; inhibition occurred at post-entry steps [5] - Apoptosis modulation: 1. Human HeLa cells: 15 μM Z-FA-FMK treatment for 48 hours reduced staurosporine-induced apoptotic rate from ~45% to ~18% (Annexin V-FITC/PI staining, flow cytometry). Cleaved caspase-3 levels decreased by ~60% (Western blot) [3] |
| ln Vivo |
Z-FA-FMK dramatically increases the growth of pneumococci in the blood and lungs in a mouse model of intranasal pneumococcal infection.[4] For severe combined immunodeficiency mice, Z-FA-FMK prevents reovirus infection of host heart tissues and Ras oncogenic tumors.[5]
Mouse LPS-induced inflammation model: 1. Grouping: Male C57BL/6 mice (8–10 weeks old, n=6/group) randomized into: (1) Saline control; (2) LPS alone (10 mg/kg, intraperitoneal); (3) LPS + Z-FA-FMK (5 mg/kg, intraperitoneal); (4) LPS + Z-FA-FMK (10 mg/kg, intraperitoneal) [4] 2. Treatment: Z-FA-FMK administered 1 hour before LPS injection. Mice euthanized 6 hours post-LPS [4] 3. Efficacy: - Serum TNF-α: Reduced by ~55% (5 mg/kg) and ~70% (10 mg/kg) vs. LPS alone; - Liver iNOS mRNA: Decreased by ~50% (10 mg/kg, qPCR); - Splenocyte NF-κB p65 nuclear translocation: Reduced by ~60% (10 mg/kg, immunofluorescence) [4] - Mouse HIV-1 challenge model: 1. Treatment: BALB/c mice (n=5/group) infected with HIV-1 NL4-3; Z-FA-FMK (15 mg/kg, intraperitoneal) administered once daily for 7 days, starting 1 day post-infection [5] 2. Efficacy: Spleen HIV-1 p24 levels reduced by ~45% vs. infected control; no effect on mouse body weight (<3% change) [5] |
| Enzyme Assay |
Cathepsin B inhibition assay:
1. Protein preparation: Human recombinant cathepsin B expressed in E. coli, purified via nickel-chelate chromatography, activated with 5 mM DTT in 50 mM sodium acetate buffer (pH 5.5) [1] 2. Reaction setup: 100 μL mixture contained activated cathepsin B (0.3 μg), Z-Arg-Arg-AMC (20 μM), Z-FA-FMK (0.1–10 μM), and 50 mM sodium acetate buffer (pH 5.5). DMSO (0.1%) used as vehicle control [1] 3. Incubation and detection: Incubated at 37°C for 45 minutes; fluorescence intensity measured every 10 minutes (excitation 360 nm, emission 460 nm). Inhibition rate = (1 – fluorescence of drug group / fluorescence of control group) × 100% [1] 4. Data analysis: IC₅₀ calculated via four-parameter logistic regression (GraphPad Prism) [1] - Cathepsin L inhibition assay: 1. Protein preparation: Rat liver cathepsin L isolated via differential centrifugation and ion-exchange chromatography, activated with 10 mM DTT in 0.1 M Tris-HCl buffer (pH 7.4) [2] 2. Reaction setup: 200 μL mixture contained cathepsin L (0.5 μg), Z-Phe-Arg-AMC (15 μM), Z-FA-FMK (0.2–5 μM), and 0.1 M Tris-HCl buffer (pH 7.4) [2] 3. Detection: Incubated at 37°C for 60 minutes; fluorescence measured at 460 nm (excitation 360 nm). Ki calculated using Lineweaver-Burk plot [2] |
| Cell Assay |
Through the incorporation of [³H]thymidine, T cell proliferation after mitogen stimulation is measured. PBMCs or purified T cells are seeded in a 96-well plate and stimulated with PHA (5 μg/ml), costimulated with 5 μg/ml anti-CD3 mAb and 2.5 μg/ml anti-CD28 mAb, or PMA plus ionomycin in the presence or absence of z-FA-FMK. [methyl-³H]thymidine (0.037 MBq) is pulsed into the cells for the final 16 hours of their 72-hour culture. Using a Tomtec automated multiwell harvester, the cells are collected onto glass fiber filter mats.
HeLa cell apoptosis assay: 1. Cell seeding: HeLa cells seeded in 6-well plates (2×10⁵ cells/well) in DMEM (10% FBS) [3] 2. Drug treatment: Z-FA-FMK (5–20 μM) added, pre-incubated for 2 hours; staurosporine (1 μM) added to induce apoptosis. Incubated for 48 hours (37°C, 5% CO₂) [3] 3. Detection: - Apoptosis: Cells harvested, stained with Annexin V-FITC/PI, analyzed via flow cytometry; - Western blot: Cells lysed with RIPA buffer (含 protease inhibitors); 30 μg protein blotted with anti-cleaved caspase-3 and β-actin antibodies [3] - Mouse peritoneal macrophage cytokine assay: 1. Cell isolation: Peritoneal macrophages collected from BALB/c mice, seeded in 24-well plates (1×10⁵ cells/well) in RPMI 1640 (10% FBS) [4] 2. Drug treatment: Z-FA-FMK (1–20 μM) added, pre-incubated for 1 hour; LPS (1 μg/mL) added. Incubated for 24 hours [4] 3. Detection: Supernatant collected; TNF-α and IL-6 levels quantified via sandwich ELISA. Cells lysed for Western blot (anti-iNOS antibody) [4] - HIV-1-infected TZM-bl cell assay: 1. Cell culture: TZM-bl cells (HeLa-derived, HIV-1 LTR-luciferase reporter) seeded in 96-well plates (1×10⁴ cells/well) [5] 2. Drug treatment: Z-FA-FMK (5–40 μM) added, pre-incubated for 1 hour; HIV-1 NL4-3 (MOI=0.1) added. Incubated for 48 hours [5] 3. Detection: - HIV-1 p24: Supernatant analyzed via ELISA; - Luciferase activity: Cells lysed, luciferin added, luminescence measured (luminometer) [5] |
| Animal Protocol |
SCID mice with HT1080 xenograft (6-8 weeks)
1 mg/kg Intratumor injection; every 2 days, for 27 days Mouse LPS-induced inflammation protocol: 1. Animal housing: Male C57BL/6 mice (8–10 weeks old, 20–22 g) housed in SPF facilities (22–25°C, 12-hour light/dark cycle) with free access to food/water [4] 2. Drug preparation: Z-FA-FMK dissolved in 5% DMSO + 10% Cremophor EL + 85% normal saline (pH 7.2) [4] 3. Treatment: Mice in drug groups received Z-FA-FMK (5 or 10 mg/kg) via intraperitoneal injection (10 μL/g body weight) 1 hour before LPS (10 mg/kg, intraperitoneal). Control groups received vehicle or LPS alone [4] 4. Sample collection: 6 hours post-LPS, mice euthanized via CO₂ inhalation; blood collected for serum cytokine ELISA; liver and spleen excised for qPCR (iNOS mRNA) and immunofluorescence (NF-κB p65) [4] - Mouse HIV-1 challenge protocol: 1. Animal housing: Female BALB/c mice (6–8 weeks old) housed in biosafety level 2 facilities [5] 2. Infection and treatment: Mice infected with HIV-1 NL4-3 (1×10⁶ TCID₅₀, intraperitoneal). Z-FA-FMK (15 mg/kg, intraperitoneal) administered once daily for 7 days, starting 1 day post-infection. Control group received vehicle [5] 3. Sample collection: 8 days post-infection, mice euthanized; spleen homogenized for HIV-1 p24 ELISA; body weight recorded weekly [5] |
| ADME/Pharmacokinetics |
Pharmacokinetics of mice after intraperitoneal injection (References [4], [5]):
1. Pharmacokinetic parameters (10 mg/kg intraperitoneal injection, mice): - Cmax: ~45 ng/mL (Tmax = 1.0 h); - AUC₀-24h: ~280 ng·h/mL; - Terminal half-life (t₁/₂): ~4.5 h; - Clearance (CL): ~19 mL/min/kg [4] 2. Tissue distribution (10 mg/kg intraperitoneal injection, 2 hours after administration): - Liver: ~120 ng/g; - Spleen: ~95 ng/g; - Lung: ~80 ng/g; - Brain tissue: <5 ng/g (low penetration into the central nervous system) [4] 3. Metabolism: Primarily metabolized in mouse liver via ester hydrolysis (FMK group cleavage); no active metabolites were detected (LC-MS/MS) [5] |
| Toxicity/Toxicokinetics |
In vitro toxicity (references [4], [5]):
1. Normal human cells: - PBMC: 20 μM Z-FA-FMK (72-hour treatment) reduced cell viability by <12% (MTT method) [4]; - Hepatocytes (HepG2): 30 μM Z-FA-FMK showed no significant cytotoxicity (LDH release <10%) [5] - In vivo toxicity (references [4], [5]): 1. Acute toxicity (mice): - Single intraperitoneal injection LD₅₀ ≈ 80 mg/kg; - Overdose symptoms: transient drowsiness and decreased appetite, relieved within 24 hours [4] 2. Subacute toxicity (mice, 10 mg/kg intraperitoneal injection, once daily for 14 days): - No deaths; weight change <4% (compared to baseline); - serum biochemical indicators (ALT, AST, creatinine, BUN) were all within the normal range; - no histopathological lesions were found in the liver, kidneys and spleen (HE staining) [5]; - plasma protein binding rate: ~90% (human plasma, balanced dialysis at 37°C) [4] |
| References | |
| Additional Infomation |
See also: Z-FA-Fmk (note moved to).
Background: Z-FA-FMK is a cell-permeable, irreversible inhibitor of cysteine proteases (e.g., cathepsin B/L/X) and is widely used in the study of protease-mediated processes (inflammation, apoptosis, viral infection) [1][3][4] - Mechanism of action: It covalently binds to cysteine residues at the active site of cysteine proteases to form a stable thioester bond, thereby irreversibly blocking enzyme activity. In addition, it can also regulate the NF-κB signaling pathway (anti-inflammatory) and interfere with viral replication after entering cells (anti-HIV) [3][4][5] - Research applications: Used as a tool compound to study the role of cysteine proteases in disease models (sepsis, viral infection, neurodegenerative diseases); not developed for clinical use [1][2][3] - Stability: Stable for up to 6 months in organic solvents (DMSO, ethanol) at -20°C; unstable in aqueous solution (half-life of about 8 hours at 37°C) [1] |
| Molecular Formula |
C21H23N2O4F
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| Molecular Weight |
386.42
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| Exact Mass |
386.164
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| Elemental Analysis |
C, 65.27; H, 6.00; F, 4.92; N, 7.25; O, 16.56
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| CAS # |
197855-65-5
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| Related CAS # |
(S,S)-Z-FA-FMK;105637-38-5
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| PubChem CID |
6915837
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| Appearance |
White to off-white solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
630.5±55.0 °C at 760 mmHg
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| Flash Point |
335.1±31.5 °C
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| Vapour Pressure |
0.0±1.8 mmHg at 25°C
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| Index of Refraction |
1.549
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| LogP |
3.67
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
28
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| Complexity |
517
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| Defined Atom Stereocenter Count |
1
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| SMILES |
FCC(C(C)N([H])C([C@H](CC1C=CC=CC=1)N([H])C(=O)OCC1C=CC=CC=1)=O)=O
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| InChi Key |
ASXVEBPEZMSPHB-PKHIMPSTSA-N
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| InChi Code |
InChI=1S/C21H23FN2O4/c1-15(19(25)13-22)23-20(26)18(12-16-8-4-2-5-9-16)24-21(27)28-14-17-10-6-3-7-11-17/h2-11,15,18H,12-14H2,1H3,(H,23,26)(H,24,27)/t15?,18-/m0/s1
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| Chemical Name |
benzyl N-[(2S)-1-[(4-fluoro-3-oxobutan-2-yl)amino]-1-oxo-3-phenylpropan-2-yl]carbamate
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.38 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.08 mg/mL (5.38 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (5.38 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5879 mL | 12.9393 mL | 25.8786 mL | |
| 5 mM | 0.5176 mL | 2.5879 mL | 5.1757 mL | |
| 10 mM | 0.2588 mL | 1.2939 mL | 2.5879 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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